Use of toll-like receptor-9 agonists

ABSTRACT

The present invention relates to the use of a TLR9 agonist and/or a TLR4 antagonist and/or a NOD2 agonist for treatment or prevention of disorders involving TLR4 activation, such as systemic sepsis and necrotizing enterocolitis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. Ser. No. 12/104,816,filed Apr. 17, 2008, which claims priority to U.S. ProvisionalApplication Ser. No. 61/027,728, filed Feb. 11, 2008, and to U.S.Provisional Application Ser. No. 60/912,862, filed Apr. 19, 2007. Thisapplication also claims priority to U.S. Provisional Application Ser.No. 61/334,999, filed May 14, 2010. Each of the foregoing applicationsare hereby incorporated by reference in their entireties.

GRANT INFORMATION

The TLR4 and TLR9-related subject matter of this application was madewith government support under Grant No. R01-GM078238-01 awarded by theNational Institutes of Health. The United States Government has certainrights in the invention. No federal funds were used in the developmentof subject matter related to NOD2.

1. INTRODUCTION

The present invention relates to the use of a Toll-like receptor-9(TLR9) agonist and/or a Toll-like receptor-4 (TLR4) antagonist and/or aNuclear Oligomerization Domain-2 (NOD2) agonist for treatment orprevention of disorders involving Toll-like receptor-4 (TLR4)activation, such as systemic sepsis and necrotizing enterocolitis. It isbased, at least in part, on the discovery that a TLR9 agonist, a TLR4antagonist, and a NOD2 agonist can suppress the consequences of TLR4activation in such conditions.

SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listingsubmitted herewith via EFS on Dec. 5, 2011. Pursuant to 37 C.F.R.§1.52(e)(5), the Sequence Listing text file, identified as0723960438SeqUst.txt, is 4,484 bytes and was created on Dec. 5, 2011.The Sequence Listing, electronically filed herewith, does not extendbeyond the scope of the specification and thus does not contain newmatter.

2. BACKGROUND OF THE INVENTION 2.1 Necrotizing Enterocolitis

Necrotizing enterocolitis (“NEC”) is the most common—and mostlethal—disease affecting the gastrointestinal tract of prematureinfants. It has become more common as the survival rate of prematureinfants has improved, and is diagnosed at an incidence of between 0.09and 0.24 percent of live births (Feng et al., 2005, Semin. Pediatr.Surg. 14:167-174; Henry et al., 2005, Semin. Pediatr. Surg. 14: 181-190;Warner et al., 2005, Semin. Pediatr. Surg. 14: 181-190; Hsueh et al.,2003, Pediatr. Dev. Pathol. 6: 6-23). Risk factors for NEC include (inaddition to prematurity), aggressive enteral feeding, episodes of birthasphyxia, polycythemia, umbilical vessel catheterization, congenitalheart disease, hyperosmolar nutritional formulas, maternal cocaine use,respiratory distress syndrome, and maternal preeclampsia (Anand et al.,2007, Shock 27(2):124-133, citing Hsueh et al., 2003, Pediatr. Dev.Pathol. 6: 6-23; Neu, 1996, Pediatr. Clin. N. Am. 43: 409-432; Kosloske,1994, Acta Pediatr. Suppl. 396:2-7; Neu et al., 2005, Semin. Pediatr.Surg. 14: 137-144; Shin et al., 2000, J. Pediatr. Surg. 35: 173-176; Ng,2001, J. Paediatr Child Health 37:1-4). In more advanced instances ofthe disease, it may result in intestinal necrosis and perforation,multisystem organ failure, systemic sepsis, and death.

Evidence suggests that the pathogenesis of NEC involves aberrantbacterial-enterocyte signaling. A role for gram negative bacteria in thepathogenesis of NEC is supported by the observations that NEC casesoften occur in epidemic outbreaks, NEC responds to systemic antibiotics,patients with NEC are frequently found to have positive blood culturesfor enteric organisms, and there are markedly increased serum levels oflipopolysaccharide (“LPS”) in patients with NEC. It has beenhypothesized that an episode of systemic stress leads to translocationof bacteria across the intestinal barrier, to result in activation ofstress pathways and of the immune system, resulting in a globalinflammatory response and tissue injury (Anand et al., 2007, Shock27(2):124-133).

Treatment of NEC involves, first, supportive therapy in the form ofnasogastric decompression and resuscitation with isotonic solutions. Inaddition, broad spectrum antibiotics are administered. More severe casesare further managed with operative intervention, including removal ofnecrotic intestine and creation of stomas. The mortality associated withNEC, particularly if intestinal perforation has occurred, is high, andhas been set at between 20 and 50 percent (Henry and Moss, 2006, NeoRev.7(9): e456). In infants having a birth weight of less than 1500 g, witha perforated intestine, despite treatment a mortality of approximately35 percent was recently observed (Moss et al., 2006, N. Engl. J. Med.354:2225-2234).

2.2 TLRs

Bacterial signaling occurs via Toll-like receptors (“TLRs”) in theintestine. TLRs participate in what is referred to as the “innate immuneresponse” and play both activating and inhibitory roles.

Gram negative bacteria and their products are known to interact withTLR4 and TLR9. TLR4, which is activated by LPS, has been reported to beexpressed on the apical surface of enterocytes and to bind andinternalize purified endotoxin (Cetin et al., 2004, J. Biol. Chem.279:24592-24600; Cario et al., 2000, J. Immunol. 164:966-972; Otte etal., 2004, Gastroenterol. 126:1054-1070). TLR 4 has also been implicatedin phagocytosis and translocation of bacteria across the intestinalbarrier (Neal et al., 2006, J. Immunol. 176:3070-3079). TLR9 has beenreported to be expressed on the colonic apical surface in wildtype, butnot germ-free, mice, suggesting that expression of TLR9 in these cellsmay be upregulated in response to pathogenic bacterial DNA (Ewaschuk etal., 2007, Inf. & Immun., published online ahead of print, doi:10.1128/IAI.01662-06).

Activating TLR9 ligands, CpG oligonucletodies (CpG ODNs) are disclosedas useful in treating inflammatory bowel disease (see U.S. Pat. No.6,613,751, Lee et al., 2006, Ann. N.Y. Acad. Sci. 1072:351-355; Katakuraet al., 2005, J. Clin. Invest. 115:695) and in lipopolysaccharide(LPS)-associated disorders (see U.S. Pat. No. 6,214,806). However, theassociation, according to the present invention, between TLR9 activationand TLR4 inhibition had not hitherto been made, nor had the use of TLR9activation in the treatment of necrotizing enterocolitis been known.

2.3 NOD2

A novel arm of the enterocyte innate immune system governed bynucleotide oligomerization domain-2 (NOD2) has recently been identified.NOD2 is a member of the NOD Like Receptors (NLR) family of cytoplasmicpathogen recognition receptors that detect bacterial motifs, inparticular the bacterial cell wall component muramyl-di-peptide(MDP)(Kanneganti and Núñez, 2008, Immunity 27:549-559). The importanceof NOD2 signaling and the development of intestinal inflammation wasconfirmed as mutations in the NOD2 gene were found to be increased in alarge cohort of patients with Crohn's disease, a chronic intestinalinflammatory disorder (Carneiro et al., 2008, J. Pathol. 214:136-148;Franchi et al., 2008, Cell Microbiol 10:1-8).

3. SUMMARY OF THE INVENTION

The present invention relates to the use of a TLR9 agonist and/or a TLR4antagonist and/or a NOD2 agonist for treatment or prevention ofdisorders involving TLR4 activation, such as systemic sepsis and NEC.The TLR9-related aspect of the present invention is based, at least inpart, on the discovery that activation of TLR9 inhibited TLR4 signalingin enterocytes in vitro and in vivo, leading to a reduction in indiciaof inflammation. The TLR4-related aspect of the present invention isbased, at least in part, on the discovery that NFκB activation,inhibited by a TLR9 agonist, could be further inhibited by a TLR4antagonist. The NOD2-related aspect of the present invention is based,at least in part, on the discovery that (i) experimental and human NECare associated with the loss of NOD2 expression in the intestinalmucosa, (ii) activation of NOD2 with the specific agonistmuramyl-di-peptide (MDP) led to a reduction in TLR4-mediated signalingin enterocytes, and (iii) administration of MDP to newborn mice in anexperimental model of NEC conferred significant protection against thedevelopment of NEC.

Accordingly, the present invention provides for methods and compositionsfor treating or preventing disorders associated with TLR4 activation, inparticular disorders epidemiologically linked to bacterial endotoxin, byadministering an effective amount of an agonist of TLR9 and/or aneffective amount of an agonist of NOD2. In a subset of non-limitingembodiments, one or more agonist of TLR9 and/or one or more agonist ofNOD2 may be administered together with an antagonist of TLR4.

In specific, non-limiting embodiments, the present invention providesfor methods comprising administering, to an infant (for example, apremature infant or a term infant otherwise at risk for the disease), aneffective amount of an agonist of NOD2, such as but not limited tomuramyl-di-peptide, which reduces the risk of the infant developing NEC.Such methods may further comprise administering an effective amount ofan agonist of TLR9 and/or an antagonist of TLR4. In related embodiments,the present invention provides for pharmaceutical compositions,including nutritional formulations, comprising effective concentrationsof NOD2 agonist which optionally further comprise one or more TLR9agonist and/or one or more TLR4 antagonist.

In further specific, non-limiting embodiments, the present inventionprovides for methods of treating NEC, including reducing the severity ofNEC, in an infant suffering from the disease, comprising administeringto the infant an effective amount of an agonist of NOD2, such as but notlimited to muramyl-di-peptide. Such methods may further compriseadministering an effective amount of an agonist of TLR9 and/or anantagonist of TLR4.

4. BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of the patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIG. 1. Western blot showing expression of TLR4 and TLR9 in positivecontrol cells and enterocytes.

FIG. 2A-B. TLR4 and TLR9 are expressed on the murine intestine. (A)Western blot showing expression of TLR4 and TLR9 in positive controlcells and in murine ileal mucosal scrapings. (B) Immunofluorescencestudies showing expression of TLR4 and TLR9 in murine intestine.

FIG. 3. Immunofluorescence studies showing expression of TLR4 and TLR9in the intestine of human neonates.

FIG. 4. Schematic diagram of a model for the etiology of NEC, where, inthe context of physiologic stressors such as hypoxia, infection, and/orprematurity, bacterial DNA and endotoxin from lumenal bacteria canactivate TLR4 as well as suppressor pathways involving TLR9.

FIG. 5. Schematic diagram of the various molecules involved in the(indirect) interactions between TLR9 and TLR4 which may be used tomeasure the effects of TLR9 on TLR4 signaling.

FIG. 6A-C. LPS signaling is attenuated by the TLR9 ligand CpG-DNA inenterocytes. (A) Comparison of phosphorylated p38 versus total p38 in anegative control, in the presence of 50 μg/ml LPS, in the presence of 50μg/ml LPS and 10 μM CpG, and in the presence of 10 μM CpG. (B)Comparison of phosphorylated ERK versus total ERK in a negative control,in the presence of LPS, in the presence of LPS and CpG, and in thepresence of CpG. (C) Bar graph showing the results of A.

FIG. 7A-E. LPS-mediated translocation in enterocytes which are either(A) untreated or treated with (B) 50 μg/ml LPS; (C) 50 μg/ml LPS+10 μMCpG; or (D) 10 μM CpG. (E) Bar graph showing the nuclear:cytoplasmicratio for NF-κB in (A)-(D).

FIG. 8. CpG-DNA reduces LPS-mediated cytokine release from enterocytes.Bar graph showing the level of supernatant IL-6 in enterocytes whichwere either untreated (“CTRL”) or treated with 50 μg/ml LPS, 50 m/mlLPS+10 μM CpG, or 10 μM CpG.

FIG. 9A-F. CpG-DNA reduces TLR4 signaling through TLR9 in enterocytes.(A) Western blot showing the amounts of TLR9 protein relative to actinin untreated enterocytes (Ctrl) or in enterocytes treated with 0.084 μMnon-specific siRNA or siRNA specific for TLR9. (B)-(E) areimmunofluorescence studies showing the relative amounts of p65 in theenterocyte nucleus versus its cytoplasm, when either (B) untreated ortreated with (C) 50 μg/ml LPS; (D) 50 μg/ml LPS+10 μM CpG (in thecontext of normal TLR9 levels) or (E) 50 μg/ml LPS+10 μM CpG (in thecontext of TLR9 knockdown by siRNA). (E) Bar graph showing the resultsof (B)-(E).

FIG. 10. Experimental design to assess whether TLR9 activation affectsTLR4-mediated inflammation in vivo.

FIG. 11A-B. Experiments according to the design shown in FIG. 10 showedthat LPS-dependent signaling and inflammation were attenuated by CpG-DNAin the murine intestinal mucosa. (A). Western blot showing levels ofsignaling molecules phospho-p38 and phospho-ERK (relative to actin) inmice which were either untreated (Ctrl) or treated with LPS, LPS+CpG, orCpG. (B) Bar graph showing serum levels of the inflammatory cytokineIL-6 in mice which were either untreated (Ctrl) or treated with LPS,LPS+CpG, or CpG.

FIG. 12. TLR4 and TLR9 expression are unchanged by CpG-DNA and LPS inenterocytes. Western blot showing levels of TLR4 and TLR9 (relative toactin) in enterocytes which were either untreated (Ctrl) or treated withLPS, LPS+CpG, or CpG.

FIG. 13A-E. CpG-DNA causes a redistribution of TLR4 into internalstructures in IEC-6 cells. Immunofluorescence studies showing TLR4distribution in IEC-6 cells which were either untreated (A) or treatedwith (B) LPS; (C) LPS+CpG; or (D) CpG. (E) is a schematic drawingshowing redistribution of TLR4 caused by TLR9.

FIG. 14A-D. LPS causes the internalization of TLR9, which is reversed byCpG-DNA. Immunofluoresence studies of IEC-6 cells showing TLR9distribution in cells which were either (A) untreated; or treated with(B) LPS; (C) LPS+CpG; or (D) CpG.

FIG. 15. Schematic drawing showing development of a model system fornecrotizing enterocolitis in the mouse.

FIG. 16. Western blot showing expression of TLR4 and TLR9 in controlmice and mice modeling necrotizing enterocolitis (“NEC”), where the micewere produced according to the protocol diagramed in FIG. 15.

FIG. 17A-D. Gross and histologic anatomies of intestines, stressed byhypoxia, of normal versus TLR4 mutant mice. (A) Intestine of a TLR4wildtype mouse, stressed by hypoxia, modeling necrotizing enterocolitis;(B) Intestine of a TLR4 mutant mouse, stressed by hypoxia; (C)histologic section from an intestine as depicted in (A); and (D)histologic section from an intestine as depicted in (B).

FIG. 18A-D. Histology of intestines from mice that were (A) breast fed(control); (B) gavage fed+hypoxic to model necrotizing enterocolitis(NEC); (C) breast fed and treated with 500 μg/kg (approximately 10μg/animal, intraperitoneally injected) CpG (control); (D) gavagefed+hypoxic to model NEC and treated with 500 μg/kg (approximately 10μg/animal, intraperitoneally injected) CpG.

FIG. 19A-H. (A-C) Immunohistochemical staining from murine terminalileum showing actin bordering villi (indicated by large arrowheads) andcaspase 3 (demonstrating apoptosis, indicated by small arrowheads) in amurine model of endotoxemia. (A) Control mice (injected with saline).(B) Mice injected with 5 mg/kg of LPS. (C) Mice injected with 5 mg/kg ofLPS and 1 mg/kg of CpG. (D-G) Immunohistochemical staining of terminalileum from newborn mice that were either breast fed (“control”) orinduced to develop NEC (“NEC”). Sections were stained for caspase 3 as amarker of apoptosis and enterocyte loss; positive staining is indicatedby a small arrowhead. (D) Control injected with saline. (E) NEC injectedwith saline. (F) Control injected with 1 mg/kg CpG-DNA daily for fourdays. (G) NEC injected with 1 mg/kg CpG-DNA daily for four days. (H) Bargraph summarizing results, showing percent apoptosis in terminal ileumas depicted in (D)-(G).

FIG. 20A-I. Newborn mice were either breast fed (“control”) or wereinduced to develop NEC (“NEC”). (A) Gross micrograph of intestine of acontrol mouse. (B) Gross micrograph of intestine of a control mousetreated with 1 mg/kg CpG-DNA daily for four days. (C) Gross micrographof intestine of a NEC mouse. (D) Gross micrograph of intestine of a NECmouse treated with 1 mg/kg CpG-DNA daily for four days. (E) Micrographof histological section of intestine of a control mouse. (F) Micrographof histological section of intestine of a control mouse treated withCpG. (G) Micrograph of histological section of intestine of a NEC mouse.(H) Micrograph of histological section of intestine of a NEC mousetreated with CpG. (I) Summary bar graph of the above results.

FIG. 21. Nuclear:cytoplasmic ratio of NFκB, indicating the extent oftranslocation of NFκB into the nucleus, in IEC-6 cells which were eitheruntreated (control); treated with LPS; treated with LPS and CpG; treatedwith LPS, CpG and polymixin B; treated with CpG; or treated withpolymixin B. The concentrations used were LPS at 50 CpG-DNA at 1 μM, andpolymixin B at 10 μg/ml.

FIG. 22A-I. NOD2 expression is reduced in human and experimental NEC.A-D: Experimental NEC was induced in newborn mice using a combination offormula gavage and hypoxia while control mice remained breast fed bytheir mothers. A. Gross appearance of terminal ileum from breast fedmice. B: Histological appearance of the terminal ileum from breast fedmice. C. Gross appearance of the terminal ileum from mice withexperimental NEC. D. Histological appearance of the terminal ileum frommice with experimental NEC. E. Gross and appearance of NEC in a preterminfant at the time of surgery. F. Microscopic appearance of NEC in apreterm infant at the time of surgery. G: Measurement of serum IL-6 inanimals with NEC compared with control animals. H. Severity of NECinduced in wild-type and TLR-mutant animals demonstrating reduction inNEC in TLR4-mutant mice. I. Real-time PCR showing the expression of NOD2in the intestine of mice (upper blot) and infants (lower blot) with NECas compared with controls without NEC. Representative of 6 separateexperiments. *p<0.05 vs. control by ANOVA.

FIG. 23A-E. MDP prevents TLR4-mediated NFkB translocation in IEC-6enterocytes. A. Confocal microscopic image demonstrating theimmunolocalization of NFkB (p65 subunit) in untreated IEC-6 cells,revealing a cytoplasmic appearance; B. LPS treatment causes a nucleardistribution of NFkB, indicating that nuclear translocation hasoccurred; C. Pre-treatment of IEC-6 cells with MDP maintains acytoplasmic appearance of NFkB, indicating that MDP limits TLR4-mediatedNFkB translocation; D. Cytoplasmic appearance of NFkB in IEC-6 cellstreated with MDP alone, indicating that MDP has minimal stimulatoryeffects on NFkB translocation in IEC-6 cells; E. Quantification of NFkBtranslocation in IEC-6 cells in the conditions indicated. Representativeof 5 separate experiments. **p<0.05 vs. control by ANOVA, *p<0.05 vs.LPS by ANOVA.

FIG. 24A-C. MDP inhibits TLR4 signaling in enterocytes. A. IL-6 releasein vitro as determined by ELISA in IEC-6 cells that were eitheruntreated, or treated with LPS in the presence or absence of MDP. B.Serum IL-6 release in vivo in wild-type (open bars) and NOD2-deficientmice (solid bars) that were either pre-treated with saline or MDP theninjected with LPS. C. SDS-PAGE showing the expression of phosphorylatedERK and beta-actin in IEC-6 cells that were treated with media(control), LPS, LPS with MDP, LPS with MDP-C which is a non-stimulatoryanalogue of MDP, and either MDP or MDP-C alone. **p<0.05 vs. control byANOVA, *p<0.05 vs. LPS by ANOVA.

FIG. 25. MDP treatment decreases TLR4 expression in enterocytes. Newbornmice were treated with LPS after pre-treatment with either saline orMDP. Three hours later mucosal scrapings were harvested from theterminal ileum, and subjected to RT-PCR for expression of TLR4.Representative of 4 separate experiments. Duplicate samples are shownfor each group.

FIG. 26A-E. MDP prevents against the development of experimental NEC invivo in newborn mice. Newborn mice were injected with saline or MDPdaily for four days, and then were induced to develop NEC. A. Histology(H&E) of terminal ileum of breast fed mouse treated with saline control.B. Histology (H&E) of terminal ileum of breast fed mouse treated withMDP. C. Histology (H&E) of terminal ileum of mouse pre-treated withsaline, after which NEC was induced. D. Histology (H&E) of terminalileum of mouse pre-treated with MDP, after which conditions whichinduced NEC in saline-treated mice were applied. E: Severity of NEC innewborn mice as scored by a blinded pediatric pathologist. *p<0.05 vs.NEC in saline-injected mice. Representative of three separateexperiments with over 5 animals per group.

FIG. 27A-G. CpG-ODN-HS and CpG-DNA reduce the development ofexperimental NEC in vivo in newborn mice. Newborn NEC and control micewere injected with CpG-ODN-HS or CpG-DNA. A. Histology (H&E) of terminalileum of breast fed mouse. B. Histology (H&E) of terminal ileum of mouseafter induction of NEC. C. Histology (H&E) of terminal ileum of mousetreated with CpG-DNA, after which conditions which induced NEC wereapplied. D. Histology (H&E) of terminal ileum of mouse treated withCpG-ODN-HS, after which conditions which induced NEC were applied. E.Histology (H&E) of terminal ileum of breast fed mouse treated withCpG-ODN-HS. F. Histology (H&E) of terminal ileum of breast fed mousetreated with CpG-DNA. G. Severity of NEC in newborn mice as scored by ablinded pediatric pathologist. *p<0.05 vs. NEC in saline-injected mice.

FIG. 28. CpG-ODN-HS and CpG-DNA reduce the expression of mucosal TNF-αin NEC newborn mice in vivo.

FIG. 29A-D. Confocal microscopy showing the extent of NFkB activation inmice with NEC in the absence or presence of CpG-ODN-HS. CpG-ODN-HSreduces the level of NFkB activation in NEC newborn mice in vivo. Themice express NFkB-GFP, and sections were stained with anti-GFP antibody(red; arrow).

FIG. 30A-C. Confocal microscopy showing the colocalization of NFkB andE-cadherin in breast fed (A), NEC (B) and NEC mice treated withCpG-ODN-HS.

5. DETAILED DESCRIPTION OF THE INVENTION

For clarity of description, and not by way of limitation, the detaileddescription of the invention is divided into the following subsections:

(i) disorders associated with TLR4 activation;

(ii) TLR9 agonists;

(iii) NOD2 agonists;

(iv) TLR4 antagonists;

(v) methods of prevention;

(vi) methods of treatment; and

(vii) pharmaceutical/nutriceutical compositions.

5.1 Disorders Associated with TLR4 Activation

TLR4-associated disorders in which endotoxin has been implicated (alsoreferred to as “endotoxin-related, TLR4-associated disorders”) (Prohinaret al., 2007, J. Biol. Chem. 282:1010-1017) include NEC (Anand et al.,2007, Shock 27:124-133) and systemic sepsis (also “sepsis,” “septicshock” or “endotoxemia”; Neal et al., 2006, J. Immunol. 176:3070-3079).Other disorders associated with TLR4 activation include, but are notlimited to, non-typeable Haemophilus influenza infection (Shuto et al.,2001, Proc. Natl. Acad. Sci. U.S.A. 98(15):8774-8779), asthma (Shan etal., 2006, Am. J. Physiol. Lung Cell Mol. Physiol. 291: L324-L333),atherosclerosis (Yang et al., 2005, Biotechnol. 42(3): 225-236), andischemic reperfusion injury (Zhai et al., 2004, J. Immunol.173:7115-7119).

5.2 TLR9 Agonists

Agonists (activators) of TLR9 which may be used according to theinvention include oligonucleotides comprising one or more unmethylatedCpG dinucleotide (“CpG ODNs”). In non-limiting embodiments of theinvention, such oligonucleotides may contain phosphorothioate linkages(at some or all bonds) or other modifications which improve stability,uptake, etc. A number of CpG ODNs that activate TLR9 are known in theart. Some are species specific.

Human CpG ODNs have been divided into three types, as follows:

-   -   Type A (D) CpG ODNs, which have polyG motifs with        phosphohorothioate linkages at the 5′ and 3′ ends and a        PO-containing palindrome CpG-containing motif at its        center—these are strong inducers of IFN-alpha production by        plasmacytoid dendritic cells and are potent NK cell activators;    -   Type B (K) CpG ODNs, which have a full phosphorothioate backbone        with one or more CpG motifs without polyG; they are potent        activators of B cells but weaker inducers of IFN-alpha        production; and    -   Type C CpG ODNs, which have a complete phosphorothioate backbone        without polyG, but have CpG motifs and palindromes; they produce        A and B-like effects (stimulate IFN-alpha and B cells).        Either type A, type B or type C human-selective CpG ODNs may be        used according to the invention, although type B CpG ODNs are        preferred. Non-limiting example of CpG ODNs which are        selectively active in humans and may be used according to the        invention include, but are not limited to, 5′-TCG TCG TTT TGT        CGT TTT GTC GTT-3′ (SEQ ID NO:1; CpG ODN 2006, InvivoGen, San        Diego, Calif.), CpG ODN 2006-G5 (InvivoGen, San Diego, Calif.),        5′-GGG GGA CGA TCG TCG GGG GG-3′ (SEQ ID NO:2; CpG ODN 2216,        InvivoGen, San Diego, Calif.), 5′-TCG TCG TCG TTC GAA CGA CGT        TGA T (SEQ ID NO:3; CpG ODN M362, InvivoGen, San Diego, Calif.),        5′-TCG TCG TTT TGT CGT TTT GTC GTT-3′ (SEQ ID NO:4; CpG ODN        7909, Coley Pharmaceutical Group, Ottawa, Ontario, Canada),        D(5′-TCTGTCGTTCT-X-TCTTGCTGTCT-5) (SEQ ID NO:5) where X is a        glycerol linker (Idera Pharmaceuticals, Cambridge, Mass.; see        Putta et al., Nucl. Acids Res. 34(11):3231-3238),        TCCATGACGTTCCTGACGTT (SEQ ID NO:6; ODN 1826, preferably        phosphorothioated), d(5′-TCTGTC*GTTCT-X-TCTTGC*TGTCT-5′) (SEQ ID        NO:7) where C*=N³-Me-dC and X is a glycerol linker (Idera        Pharmaceuticals, Cambridge, Mass.; see Putta et al., Nucl. Acids        Res. 34(11):3231-3238), and d(5′-TCTGTCG*TTCT-X-TCTTG*CTGTCT-′)        (SEQ ID NO:8) where G*=N¹-Me-dG and X is a glycerol linker        (Idera Pharmaceuticals, Cambridge, Mass.; see Putta et al.,        Nucl. Acids Res. 34(11):3231-3238).

In further embodiments, the present invention provides for the use ofCpG ODNs which are at least 90 percent and preferably at least 95percent homologous to any of the CpG ODNs referred to herein (wherehomology may be determined by standard software such as BLAST or FASTA).

In one particular, non-limiting embodiment, the CpG ODN,5′-TCCATGACGTTCCTGACGTT-3′ (SEQ ID NO:6), containing phosphorothioatelinkages, known in the art as CpG ODN 1826 (Coley Pharmaceutical Group,Ottawa, Ontario, Canada), which shows selective activation of murineTLR9, may be used. In addition, CpG ODNs which are at least about 90percent, and preferably at least about 95 percent, homologous to CpG ODN1826 may be used, where homology may be measured using a standardsoftware program such as BLAST or FASTA.

In yet another specific, non-limiting embodiment, the CpG-ODN 5′TCGTCGTTTTGTCGTTCCTGACGTT 3′ (SEQ ID NO:10), referred to herein asCpG-ODN-HS, may be used. In addition, CpG ODNs which are at least about90 percent, and preferably at least about 95 percent, homologous to CpGODN HS may be used, where homology may be measured using a standardsoftware program such as BLAST or FASTA. In non-limiting embodiments ofthe invention, a mixture of two or more CpG ODNs may be used.

In some non-limiting embodiments, the CpG ODN is at least 6, or at least7, or at least 8, or at least 9, or at least 10, or at least 11, or atleast 12, or at least 13, or at least 14, or at least 15, or at least16, or at least 17, or at least 18, or at least 19, or at least 20, orat least 21, or at least 22, or at least 23, or at least 24, or at least25, or at least 26, or at least 27, or at least 28, or at least 29, orat least 30 nucleotides in length.

In some non-limiting embodiments, the CpG-ODN is up to about 25, up toabout 30, up to about 35, up to about 40, up to about 45 or up to about50 nucleotides in length.

The CpG-ODN may optionally be linked to a carrier compound which may ormay not be a nucleic acid, for example, but not limited to, a transportpeptide that facilitates cellular uptake. The CpG-ODN may optionally becomplexed with one or more additional compound, such as a peptide, orcomprised in a micelle or liposome, to facilitate uptake.

In one non-limiting embodiment, the CpG ODN is about 15, about 16, about17, about 18, about 19, about 20, about 21, about 22, about 23, about24, about 25, about 26, about 27, about 28, about 29, about 30, about31, about 32, about 33, about 34, or about 35 nucleotides in length.

In some non-limiting embodiments, the CpG-ODN comprises the sequence 5′GTCGTT 3′.

In some non-limiting embodiments, the CpG-ODN comprises the sequence 5′GTCGTTT 3′.

In some non-limiting embodiments, the CpG-ODN comprises the sequence 5′CGTCGTTT 3′.

In some non-limiting embodiments, the CpG-ODN comprises the sequence 5′GTCGTTTT 3′.

In some non-limiting embodiments, the CpG-ODN comprises the sequence 5′CGTCGTTTT 3′.

In some non-limiting embodiments, the CpG-ODN comprises the sequence 5′GTCGTTTTGTC 3′.

In some non-limiting embodiments, the CpG-ODN comprises the sequence 5′TCGTCGTTTTGTC 3′.

In some non-limiting embodiments, the CpG-ODN comprises the sequence 5′GACGTT 3′ .

In some non-limiting embodiments, the CpG-ODN comprises the sequence 5′TGACGTT 3′.

In some non-limiting embodiments, the CpG-ODN comprises the sequence 5′CTGACGTT 3′.

In some non-limiting embodiments, the CpG-ODN comprises the sequence 5′TCCTGACGTT 3′.

In some non-limiting embodiments, the CpG-ODN comprises one or more of5′ GTCGTTTTGTC 3′, for example, one, two, three or four of 5′GTCGTTTTGTC 3′.

In some non-limiting embodiments, the CpG-ODN comprises one or more copyof 5′ GTCGTT 3′, 5′ GTCGTTT 3′, 5′ CGTCGTTT 3′, 5′ GTCGTTTT 3′, 5′CGTCGTTTT 3′ SEQ ID NO:16, SEQ ID NO:17, or a combination thereof, forexample, one, two, three or four copy or copies of 5′ GTCGTT 3′, 5′GTCGTTT 3′, 5′ CGTCGTTT 3′, 5′ GTCGTTTT 3′, 5′ CGTCGTTTT 3′ SEQ IDNO:16, SEQ ID NO:17, or a combination thereof.

In some non-limiting embodiments, the CpG-ODN comprises one or more copyof 5′ GTCGTT 3′, for example, one, two, three or four copy or copies of5′ GTCGTT 3′, and also comprises one or more copy or copies of 5′ GACGTT3′, 5′ TGACGTT 3′, 5′ CTGACGTT 3′, SEQ ID NO: 21, or a combinationthereof.

In some non-limiting embodiments, the CpG-ODN comprises one or more copyof 5′ GTCGTT 3′, 5′ GTCGTTT 3′, 5′ CGTCGTTT 3′, 5′ GTCGTTTT 3′, 5′CGTCGTTTT 3′ SEQ ID NO:16, SEQ ID NO:17, or a combination thereof, forexample, one, two, three or four copy or copies of SEQ ID NO:11, andalso comprises one or more copy or copies of 5′ GACGTT 3′, 5′ TGACGTT3′, 5′ CTGACGTT 3′, SEQ ID NO:21, or a combination thereof. In somenonlimiting embodiments, the CpG-ODN comprises 5′ GTCGTT 3′ and 5′GACGTT 3′.

For additional TLR9 agonists, see Daubenberger, 2007, Curr. Opin. Molec.Ther. 9:45-52 and Krieg, 2006, Nat. Rev. Drug Disc. 5:471-484.

In non-limiting embodiments, the present invention provides for methodsof identifying TLR9 agonists which may be used according to theinvention comprising identifying a molecule which is capable of bindingto TLR9 under physiologic conditions and which, in an in vivo system, inthe presence of a TLR4-activating amount of LPS, decreases one or moreof the relative amount of phosphorylated p38, the relative amount ofphosphorylated ERK, the relative translocation of NF-κB into thenucleus, or the amount of IL-6 produced. In addition to identifying testagents suitable for TLR9 activation, such method may also be used toconfirm the activity or optimize the dosage of any of the particular CpGODNs listed herein.

5.3 NOD2 Agonists

Any agonist (activator) of NOD2 may be used according to the invention.In specific non-limiting embodiments of the invention, the activator ofNOD2 is muramyl-di-peptide (“MDP”). MDP may be obtained, for example butnot by way of limitation, from InvivoGen (San Diego, Calif.).Alternatively, a molecule comprising MurNAc attached to L-ALa andD-isoGln other than MDP may be used. Additional non-limiting examples ofNOD2 agonists include, but are not limited to, MurNAc-L-Ala-D-isoGln,also called GM-Di; MurNAc-L-Ala-γ-D-Glu-L-Lys, also called MtriLYS; andiDAP.

5.4 TLR4 Antagonists A number of inhibitors/antagonists of TLR4 whichmay be used according to the invention (in a subset of embodiments)include, but are not limited to, LPS antagonists, for example, thefollowing:

LPS from E. coli K12 msbB (InvivoGen, San Diego, Calif.);

polymyxin B (polymixin B; polymyxin B sulfate);

CyP (Macagno et al., 2006, J. Exp. Med. 203(6):1481-1492);

lipid IVa;

E5531 (Kobasyashi et al., 1998, Antimic. Ag. Chemother.42(11):2824-2829); and

E5564 (eritoran, Eisai Co., Tokyo, Japan; Mullarkey et al., 2003, J.Pharm. Exp. Ther. 304(3): 1093-10102; Rossignol et al., 2004,Antimicrob. Agents Chemother. 48(9):3233-3240).

In a further non-limiting embodiment of the invention, an antibody(including conventional immunoglobulin, single-chain antibody, a Fabfragment, a Fv fragment, a single-chain Fv fragment, etc.) thatantagonizes TLR4 activity may be used. Such an antibody may be preparedusing standard techniques. The ability of such an antibody to act as anantagonist of TLR4 may be confirmed by the ability of the antibody toblock a LPS induced index of TLR4 activation, such as an increase inrelative phosphorylation of p38 or ERK or an increase in IL-6.

5.5 Methods of Prevention

In specific, non-limiting embodiments, the present invention providesfor methods of preventing NEC in an infant (for example, a prematureinfant or a term infant otherwise at risk for the disease) comprisingadministering, to the infant, an effective amount of an agonist of NOD2,such as but not limited to muramyl-di-peptide, which reduces the risk ofthe infant developing NEC.

According to the invention, “methods of preventing” are defined asmethods which reduce the risk of developing the disease, and do notnecessarily result in 100% prevention of the disease. As such, thesemethods, applied prophylactically to an infant, may not only reduce therisk but may also reduce the severity of the disease if it does occur.By definition, such preventative methods may be administered to aninfant having no signs of preexisting NEC as well as to an infant whichis exhibiting one or more early clinical sign consistent with NEC but inwhich a definitive diagnosis of NEC has not been established.

The NOD2 agonist may be administered by any route known in the art,including oral administration, intravenous administration, andadministration directly into the intestine.

In specific, nonlimiting embodiments, the NOD2 agonist may beadministered at a dose of between about 0.1 and 10 mg/kg, or between 0.5mg/kg and 5 mg/kg. In specific, non-limiting embodiments, the NOD2agonist may be MDP administered at a dose of between about 0.1 and 10mg/kg, or between 0.5 mg/kg and 5 mg/kg, or about 1 mg/kg. The dose maybe administered at least once a day for a period of between one day andten days, or between one day and five days, or at least three days, orat least four days, or at least five days, or until the infant isdetermined to no longer be at risk for developing NEC.

Such methods may further comprise administering an effective amount ofan agonist of TLR9 and/or an antagonist of TLR4. In such methods, theTLR9 agonist and/or TLR4 antagonist may be administered together withthe NOD2 agonist or separately.

5.6 Methods of Treatment

In a first set of embodiments, the present invention provides for amethod of treating a TLR4-associated disorder in a subject comprisingadministering, to the subject, an effective amount of a TLR9 agonist. Ina subset of such embodiments, the present invention provides for furtheradministering, to the subject, an effective amount of a TLR4 antagonist.

In a related, second set of embodiments, the present invention providesfor a method of treating an endotoxin-related, TLR4-associated disorderin a subject comprising administering, to the subject, an effectiveamount of a TLR9 agonist. In a subset of such embodiments, the presentinvention provides for further administering, to the subject, aneffective amount of a TLR4 antagonist.

When a TLR9 agonist and a TLR4 antagonist are administered in the sameregimen, the effective amounts of TLR9 and TLR4 may be such that the neteffect is a decrease in indices of inflammation, whereas the amounts ofeach agent if used individually may be either effective or ineffective(in other words, the effective dose when the agents are used incombination may be lower than the effective doses of each agent usedindividually, although individually effective doses of each agent mayalso be used in combination). Accordingly, the present inventionprovides for a method of treating a TLR4-associated disorder, comprisingadministering, to a subject in need of such treatment, an effectiveamount of a TLR9 agonist and a TLR4 antagonist.

The TLR9 agonist and/or TLR4 antagonist may be administered by any routeknown in the art, including, but not limited to, intravenous,intraarterial, oral or rectal (including via an orally or rectallyinserted catheter) administration. Where both TLR9 agonist and TLR4antagonist are included in a treatment regimen, they may be administeredconcurrently or in series.

An effective amount of a TLR9 agonist is an amount which can suppressthe effect of LPS in an in vitro or in vivo system, preferably reducinga marker of inflammation, such as relative phospho-p38 expression NF-κBtranslocation to the nucleus, or IL-6 production, by at least about 10percent or at least about 20 percent. The amount may be a concentrationor a dosage in an organism. For example, but not by way of limitation,the dose range at which a TLR9 activator, such as CpG ODN, may beadministered may be between about 100 μg/kg and 10 mg/kg, or betweenabout 100 μg/kg and 1 mg/kg, or about 500 μg/kg, which may beadministered as a single dose or a divided dose.

An effective amount of a TLR4 antagonist is an amount which can suppressthe effect of LPS in an in vitro or in vivo system, preferably reducinga marker of inflammation, such as relative phospho-p38 expression NF-κBtranslocation to the nucleus, or IL-6 production, by at least about 5percent, at least about 10 percent or at least about 20 percent, or morewhen used together with a TLR9 agonist. The dose range at which TLR4inhibitors may be administered may be, for example but not by way oflimitation, as follows (in single or divided doses):

for LPS from E. coli K12 msbB (InvivoGen, San Diego, Calif.) betweenabout 100 μg/kg and 1 mg/kg;

for polymyxin B between about 1-5 mg/kg and preferably between 2-3mg/kg;

for CyP between about 30 mg/kg and 50 mg/kg;

for lipid IVa between about 100 μg/kg and 1 mg/kg;

for E5531 between about 10 μg/kg and 1 mg/kg; and

for E5564, for a human subject, between about 20 mg and 200 mg, orbetween about 40 mg and 110 mg, said dose administered in divided dosesover a period of time ranging from about 2 to 7 days, preferably betweenabout 80 and 120 mg or about 105 mg administered over a 6 day period(e.g., 11 doses administered at 12 hour intervals)(http://www.japancorp.net/Article.Asp?Art_ID=10765).

In further specific, non-limiting embodiments, the present inventionprovides for methods of reducing the severity of NEC in an infantsuffering from the disease, comprising administering to the infant aneffective amount of an agonist of NOD2, such as but not limited tomuramyl-di-peptide. In specific, nonlimiting embodiments, the NOD2agonist may be administered at a dose of between about 0.1 and 10 mg/kg,or between 0.5 mg/kg and 5 mg/kg. In specific, non-limiting embodiments,the NOD2 agonist may be MDP administered at a dose of between about 0.1and 10 mg/kg, or between 0.5 mg/kg and 5 mg/kg, or about 1 mg/kg. Thedose may be administered at least once a day for a period of between oneday and ten days, or between one day and five days, or at least threedays, or at least four days, or at least five days, or until the infantis determined to no longer be at risk for developing NEC. Such methodsmay further comprise administering an effective amount of an agonist ofTLR9 and/or an antagonist of TLR4 (as set forth above).

The methods of treatment according to the invention may further comprisethe use of other biologically active agents, for example agents whichhad hitherto been used in the art to treat the TLR4 associated disorder,but where the addition of the inventive method and/or compositionprovides substantial therapeutic benefit. For example, but not by way oflimitation, the treatment of NEC or sepsis may further include theadministration of one or more antibiotic agent.

“Treatment” according to the invention includes, without limitation, (1)decreasing the level of one or more index of inflammation (e.g.,inflammatory cytokines such as TNF-α, IL-6, IL-12p40, IL-1β; (2)decreasing a clinical marker of inflammation, such as leukocyte count,fever, hypotension; and/or (3) reducing the risk of an adverse outcome,such as death, organ failure, hypoxia, or the need for surgery.“Treatment” does not necessarily mean that the condition being treatedwill be cured.

5.7 Pharmaceutical/Nutraceutical Compositions

The present invention, in non-limiting embodiments, provides fortherapeutic compositions.

In one set of embodiments, the therapeutic composition is a kitcomprising, in separate containers, a pharmaceutical compositioncomprising an effective amount of a TLR9 agonist and a pharmaceuticalcomposition comprising an effective amount of a TLR4 antagonist.

In another set of embodiments, the therapeutic composition is apharmaceutical composition comprising an effective amount of a TLR9agonist and a TLR4 antagonist in a suitable pharmaceutical carrier.

In yet another set of embodiments, the present invention provides for apharmaceutical composition comprising an effective concentration of NOD2agonist which may optionally further comprise an effective concentrationof one or more TLR9 agonist and/or an effective concentration of one ormore TLR4 antagonist.

An effective amount or an effective concentration of a TLR9 agonist, aNOD2 agonist, or a TLR4 antagonist is a concentration which, whenadministered in a volume suitable to the chosen route of administration,results in an effective dosage as set forth above.

In a non-limiting embodiment, the present invention provides for aninfant formula (e.g., nutritional formulation) which comprises aneffective amount of an agonist of NOD2, optionally further comprising aneffective amount of an agonist of TLR9 and/or an effective amount of anantagonist of TLR4. When administered in the amount recommended fornutritional purposes, an effective dosage of NOD2 agonist and optionallyTLR9 agonist and/or TLR4 inhibitor may be administered. In a specific,non-limiting example, the NOD2 agonist is MDP.

6. EXAMPLE 1

Both TLR4 and TLR9 were demonstrated on the surface of enterocytes frommice and humans. FIG. 1 and FIG. 2A show Western blots depictingexpression of TLR4 and TLR9 in positive control cells and enterocytesfrom C57/B16. FIG. 2B shows an image from an immunofluorescence studydemonstrating expression of TLR4 and TLR9 in murine intestine. FIG. 3shows the results of a comparable immunofluorescence study performedusing intestine from human neonates.

Experiments were performed to validate a model for the etiology of NEC,where, in the context of physiologic stressors such as hypoxia,infection, and/or prematurity, bacterial DNA and endotoxin from lumenalbacteria can activate TLR4 as well as suppressor pathways involving TLR9(FIG. 4). A variety of molecules may be used to measure the activationlevel of TLR4, including MAP kinases such as p38 and ERK and NFκB or itssubunits, p65 or p50 (FIG. 5).

A first series of experiments was designed to study the consequences ofthe TLR4 activator LPS and a CpG TLR9 agonist on the mediators ofactivated TLR4, p38 and ERK. Throughout this example section, the CpGused was CpG ODN, 5′-TCCATGACGTTCCTGACGTT-3′ (SEQ ID NO:6) P38 and ERKare both phosphorylated in their activated form. As shown in FIG. 6A-C,the relative levels of phosphorylated p38 and ERK increased whenenterocytes were exposed to LPS. However, when enterocytes were exposedto both LPS and CpG, the magnitude of this increase was significantlyless (see especially FIG. 6C). These studies show that LPS signaling isattenuated by the TLR9 ligand CpG-DNA in enterocytes.

A second series of experiments was designed to study the consequences ofthe TLR4 activator LPS and the TLR9 activator CpG on the mediator ofactivated TLR4, NFκB. As shown by comparing FIGS. 7A and B, LPS causesNFκB to translocate into the nucleus. The extent of translocation causedby LPS is decreased by co-exposure to CpG (FIGS. 7C and E). Accordingly,these studies show that LPS-mediated NF-κB translocation in enterocytesis attenuated by the TLR9 agonist CpG.

A third series of experiments, depicted in FIG. 8, showed that, instudies similar to those described above, CpG-DNA reduces LPS-mediatedcytokine release from enterocytes.

All the foregoing three series of experiments have shown that CpG (anactivator of TLR9) inhibited the effects of TLR4 activation, includingits role in promoting inflammation. To test whether CpG was, in fact,acting through TLR9, “knock-down” studies were performed in whichTLR9-specific interfering RNA (siRNA) was used to reduce expression ofTLR9 (see Western blot of FIG. 9A, which shows that the level of TLR9protein was effectively suppressed). As shown in FIGS. 9E and F(relative to FIGS. 9C and D), the attenuating effect of CpG ontranslocation of NFκB subunit p65 was virtually eliminated inenterocytes in which TLR9 expression was suppressed by siRNA. Thesestudies indicate that, in enterocytes, CpG-DNA reduces TLR4 signalingthrough TLR9.

Further experiments were performed to study the mechanism by which TLR9suppresses the TLR4 activation. Surprisingly, as shown in FIG. 12, TLR4and TLR9 expression are unchanged by CpG-DNA and LPS in enterocytes.Rather, as shown in FIG. 13A-E, it appears that CpG-DNA causes aredistribution of TLR4 into internal enterocyte structures. Inparticular, the experiments showed that while LPS causes theinternalization of TLR9, this effect is reversed by CpG-DNA (FIG.14A-D).

Experiments were then designed to determine whether the above phenomena,observed in vitro, could be confirmed in vivo (see FIG. 10). As shown inFIG. 11A-B, experiments according to the design shown in FIG. 10 showedthat LPS-dependent signaling and inflammation, as measured by levels ofphosphorylated p38 and ERK and by serum IL-6 levels, were attenuated byCpG-DNA in the murine intestinal mucosa.

To determine the relevance of all the above observations to NEC, amurine model of NEC was developed, in which hypoxia was used to induce apathology comparable to NEC in newborn mice (FIG. 15). FIG. 16 presentsa Western blot showing expression of TLR4 and TLR9 in control mice andmice modeling necrotizing enterocolitis (“NEC”). A substantial increasein the level of TLR4, and a decrease in TLR9, was observed. Interesting,the NEC-like pathology could not be induced in mice carrying a TLR4mutation (FIG. 17A-D). Finally, CpG was found to limit the severity ofexperimental NEC induced in wild-type TLR4 animals (FIG. 18A-D).

7. EXAMPLE 2

Materials and Methods

Cell culture and reagents. IEC-enterocytes and J774 macrophages wereobtained from the American Type Culture Collection (ATCC, Manassas,Va.). Phosphorothioated CpG-DNA, oligodeoxynucleotide (ODN) 1826(TCCATGACGTTCCTGACGTT) (SEQ ID NO:6), and control GpC-DNA, control ODN1826 (TCCATGAGCTTCCTGAGCTT) (SEQ ID NO:9), were synthesized by theUniversity of Pittsburgh DNA synthesis facility. ODNs were confirmed tobe endotoxin-free by Limulus assay. Antibodies were obtained as follows:TLR4; TLR9-Imgenex, San Diego, Calif.; NFkB (p65 subunit)— Santa CruzBiotechnology, Santa Cruz, Calif.; cleaved caspase-3, phospho-p38-MAPK,phospho-ERK, total p38-MAPK, and total ERK—Cell Signaling Technology,Beverly, Mass.

Induction of necrotizing enterocolitis. All mice were housed and caredfor at Rangos Research Center, Children's Hospital of Pittsburgh. Allexperiments were approved by the Children's Hospital of PittsburghAnimal Care Committee and the Institutional Review Board of theUniversity of Pittsburgh (protocol 45-06). Swiss-Webster (CfW) andC57/B1-6 mice were obtained from Jackson Laboratories (JacksonLaboratory, Bar Harbor, Me.). Endotoxemia was induced in 2 week oldC57/B16 or CpG1 (TLR9-mutant) mice by the intraperitoneal injection ofLPS (Escherichia coli 0111:B4 purified by gel-filtrationchromatography, >99% pure, 5 mg/kg, Sigma-Aldrich, St. Louis, Mo.). Inparallel, mice were administered vehicle (saline) or CpG-DNA (1 mg/kg).Three hours after injection, animals were sacrificed. To induceexperimental NEC, 10-14 day-old mice (Swiss-webster, C57B1-6 orTLR9-mutant (CpG1)) were gavage fed (Similac Advanced infant formula(Ross Pediatrics): Esbilac canine milk replacer at a ratio of 2:1) fivetimes daily, and exposed to intermittent hypoxia (5% O₂, 95% N₂) for 10minutes using a modular hypoxic chamber (Billups-Rothenberg, DelMar,Calif.) twice daily for 4 days. Animals were fed 200 microliters per 5grams of mouse body weight by gavage over 2-3 minutes, using a 24-Frenchangio-catheter which was placed into the mouse esophagus under directvision. Samples were harvested at day four for analysis. It has beendemonstrated that this experimental protocol induces intestinalinflammation and the release of pro-inflammatory cytokines in a patternthat closely resembles human NEC. Control (i.e. non NEC) animalsremained with their mothers and received breast milk. Where indicated,breast fed animals of all strains were injected with CpG-DNA 1 mg/ml ata daily dose of 1 mg/kg for 4 days prior to sacrifice or were exposed tohypoxia alone. The severity of experimental NEC was graded using apreviously validated scoring system from 0 (normal) to 3 (severe). Atsacrifice, serum was obtained by retro-orbital puncture, and terminalilea was harvested in 10% neutral buffered formalin or frozen in liquidnitrogen after embedding in Cryo-Gel (Cancer Diagnostics, Inc.). Whereindicated, mucosal scrapings were obtained by microdissection under 20×power, and collected in RNAlater (Qiagen, Valencia, Calif.).

Immuno-analysis. Cells were grown and treated in 12-well plates on glasscoverslips and fixed with 4% paraformaldehyde and permeabilized with0.1% Triton X-100 (Sigma-Aldrich, St. Louis, Mo.), blocked 5% goatserum, and after immunostaining were imaged using an Olympus Fluoview1000 confocal microscope under oil-immersion objectives. Images werecropped using Adobe Photoshop CS2 software (Adobe Systems Inc., SanJose, Calif.). In parallel, Cryo-Gel (Cancer Diagnostics, Inc.) frozensections of terminal ileum were sectioned (4 μm), rehydrated with PBSand fixed with 2% paraformaldehyde. Non-specific binding was blockedwith 5% bovine serum albumin (BSA). Sections were imaged on an OlympusFluoview 1000 confocal microscope using oil immersion objectives.

Assessment of NFkB activation. IEC-6 enterocytes were treated with LPS(50 μg/ml, Sigma-Aldrich, St. Louis, Mo.) and/or CpG-DNA (1 μM) eitheralone or in combination for 1 hour and immunostained with antibodiesagainst the p65 subunit of NF-κB. Quantification of nucleartranslocation was performed as adapted from Ding, et al. J Biol. Chem.1998 Oct. 30; 273(44):28897-905. A threshold limit was set based uponthe emission signal for DRAQ5 staining, which defined a nuclear regionof interest (ROI). Symetric expansion of the nuclear ROI by 12 pixelsdefined a nuclear and cytoplasmic ROI. The emission within this ROI wassubjected to calculation of area, integrated intensity, and averageintensity using MetaMorph software version 6.1 software. The averageNF-κB p65 intensity of the cytoplasmic area was determined bysubtracting the area and integrated intensity of the nuclear ROI fromthe nuclear+cytoplasmic ROI and dividing the cytoplasmic integratedintensity by the cytoplasmic area. The extent of p65 staining in thenucleus versus the cytoplasm (i.e. the nuclear to cytoplasmic ratio) wascalculated for each cell by dividing the nuclear average NF-κB p65intensity by the cytoplasmic average NF-κB p65 intensity. Nuclear tocytoplasmic ratio was calculated for more than 200 cells per treatmentgroup for more than 4 separate experiments.

Statistical Analysis. Statistical analysis was performed using SPSS 13.0software.s ANOVA was used for comparisons for experiments involving morethat two experimental groups. Two-tailed student's t-test was used forcomparison for experiments consisting of two experimental groups. Foranalysis of NEC severity, chi-square analysis was used.

Results

CpG-DNA was found to inhibit LPS-induced enterocyte apoptosis in murinemodels of endotoxemia as well as NEC. In a murine model of endotoxemia,immunohistochemical staining of terminal ileum of mice injected witheither saline (FIG. 19A), LPS (FIG. 19B) or LPS and CpG-DNA (FIG. 19C)demonstrated that apoptosis occurring in the enterocytes of LPS-treatedanimals was substantially reduced in LPS and CpG-DNA treated animals.Similar findings were observed in mouse models of NEC (FIG. 19D-G,summarized in FIG. 19H). Administration of CpG-DNA to the NEC animalssubstantially reduced apoptosis.

FIG. 20A-I illustrates the anatomical and histologic correlates of theresults presented in FIGS. 19D-G. Gross and histological micrographs ofcontrol and NEC mice treated with CpG-DNA demonstrate a substantialinhibition of the NEC pathology in CpG-DNA treated animals, assummarized in FIG. 20I.

Further, it was found that, when LPS-induced activation of NFκB (withsubsequent translocation into the nucleus) was measured in IEC-6 cells,while CpG-DNA significantly inhibited translocation, the combination ofCpG-DNA with the TLR4 antagonist polymixin B was even more effective atinhibiting translocation (FIG. 21). This indicates that the combinationof a TLR9 agonist (e.g., CpG-DNA) with a TLR4 antagonist (polymixin B)has at least an additive effect in attenuating TLR4 signaling inenterocytes.

8. EXAMPLE 3

NOD2 expression in the intestine is reduced in human and experimentalnecrotizing enterocolitis. In order to define the molecular mechanismsthat lead to the development of NEC, a newborn mouse model of thisdisease was developed that parallels the findings seen in human NEC(Leaphart et al., 2007, J. Immunology 179:4808-4820; Leaphart et al.,2007, Gastroenterology 132:2395-2411; Cetin et al., 2007, Am J PhysiolGastrointest Liver Physiol 292:G1347-1358). As is shown in FIG. 22A-I,newborn mice were randomized to be either breast-fed (“control”, panelsA, B) or gavaged with formula (Canine-Simialac 70%, water 30%) threetimes daily and subjected to 2 minutes of hypoxia (5% O2) in a ModularIncubator Hypoxic Chamber (Billups-Rothenberg) three times daily priorto each feeding (“NEC”, panels C, D). Animals were killed on day 4 andthe distal 2 cm of terminal ileum was harvested for histological andmolecular analysis. The histological and gross appearance of the ileumin mice with experimental NEC (FIG. 22C, D) appears similar to that ofthe ileum in infants that undergo surgical resection for severe NEC(FIG. 22E, F), and serum levels of the pro-inflammatory cytokineinterleukin-6 are increased in experimental NEC (panel G) similar tothat observed in the clinical disease (Sharma et al., 2007, J PediatrSurg 42:454-461).

Utilizing the experimental model described above, the importance of TLR4signaling in the pathogenesis of NEC was defined. To do so, wild-type(C3H/HeOUJ) and TLR4-mutant mice (C3H/HeJ) mice were subjected to themodel and the severity of NEC that developed was assessed by a blindedpathologist. As shown in FIG. 22H, the severity of NEC was significantlyreduced in TLR4-mutant mice compared to wild-type littermates. Moreover,the expression of NOD2 was significantly reduced in mice with NECcompared to control mice, a similar finding to that observed in theintestine obtained from infants undergoing resection for severe NEC ascompared to the expression in “control” infants at the time of stomaclosure (FIG. 22I). Taken together, these findings indicate a criticalrole for TLR4 in the pathogenesis of NEC, and illustrate that theexpression of NOD2 is reduced in NEC in NEC in mice and humans.

NOD2 activation with MDP inhibits TLR4 signaling in enterocytes. Thenext experiments were designed to determine whether NOD2 activation withMDP would inhibit TLR4 signaling in enterocytes. To do so, IEC-6enterocytes—a cell line that represents a model system to studyenterocyte biology and which expresses TLR4 (Neal et al., 2006, JImmunol 176:3070-3079)—were treated with LPS in the presence or absenceof MDP. Since TLR4 signaling leads to the translocation of NFkB from thecytoplasm into the nucleus, the extent of TLR4 activation was evaluatedusing an immunofluorescence-based detection assay of the p65 subunit ofNFkB. As is shown in FIG. 23A and quantified in FIG. 23E, in controlcells, NFkB is localized in the cytoplasm. Upon treatment with LPS (50μg, 1 h), NFkB was detected in the nucleus, indicative of NFkBactivation (FIG. 23B). Importantly, treatment of cells with LPS in thepresence of the NOD2 agonist MDP leads to a reduction in nucleartranslocation and the persistence of NFkB in the cytoplasm (FIG. 23C).Treatment of IEC-6 cells with MDP alone did not significantly alter theextent of NFkB translocation (FIG. 23D).

The next series of experiments were designed to further define thephysiological significance of the finding that MDP reduces TLR4-mediatedNFkB translocation in enterocytes, and to evaluate potential mechanismsinvolved. Since NFkB activation is known to lead to the release ofpro-inflammatory cytokines including IL-6, experiments were performed toevaluate whether MDP would alter the extent of IL-6 release fromLPS-treated IEC-6 cells. As shown in FIG. 24A, treatment of IEC-6 cellsin vitro with LPS led to a significant increase in IL-6 release comparedwith untreated cells, that was significantly reduced upon exposure toMDP. To determine the physiological significance of this work in vivo,wild-type and NOD2-knockout mice were injected with LPS (5 mg/kg) in thepresence or absence of MDP (1 mg/kg), and serum IL-6 release—a measureof TLR4 signaling in vivo—was determined by ELISA. As is shown in FIG.24B, MDP significantly reduced the extent of LPS-mediated IL-6 releasein wild-type mice confirming a reduction in TLR4 signaling in vivo. Theeffects of MDP in reducing TLR4 signaling were less pronounced inNOD2-knockout mice, confirming the specificity of the effect of MDP forNOD2 (FIG. 24B).

To further define the effects of MDP on LPS-mediated signaling inenterocytes, IEC-6 cells were treated with LPS in the presence orabsence of MDP and the expression of the TLR4 downstream target pERK wasassessed by SDS-PAGE. As is shown in FIG. 24C, LPS caused an increase inthe expression of pERK compared with untreated cells. Strikingly,pre-treatment with MDP significantly reduced the extent of pERKphosphorylation, and returned levels to that of untreated cells.Treatment of cells with the inactive isoform of MDP (i.e. MDPC) atequimolar concentrations in the presence of LPS did not reduce theextent of pERK expression (FIG. 24C) or IL-6 release. Taken together,these findings indicate that NOD2 activation with MDP leads to aninhibition of TLR4 signaling in enterocytes in vitro and in vivo.

MDP treatment of enterocytes reduces the expression of TLR4. The nextseries of studies investigated the potential mechanisms by which MDPactivation of NOD2 could lead to a reduction in the extent of TLR4signaling. It was first determined that MDP does not alter the relativedistribution of TLR4 in enterocytes, as confirmed usingimmunohistochemistry. By contrast, MDP leads to a significant reductionin the expression of TLR4 in enterocytes (FIG. 25), suggesting apotential mechanism by which MDP could limit TLR4 signaling. Takentogether, these findings suggest a potential mechanism by which MDPactivation of NOD2 could inhibit TLR4 signaling.

MDP prevents against the development of experimental necrotizingenterocolitis. The previous experiments indicate that TLR4 plays acritical role in the pathogenesis of NEC, and that NOD2 activation withMDP inhibits TLR4 signaling in enterocytes. It was also determined thatmucosal NOD2 expression is decreased in experimental NEC (FIG. 22I).These findings suggest that MDP administration may prevent thedevelopment of NEC. To test this directly, either saline (vehicle) orMDP (1 mg/ml, with each feed) were administered to NOD2-wild-type micedaily for four days, and then NEC was induced as in FIG. 22I. As shownin the histological sections obtained from the terminal ilea, salinetreated mice developed severe NEC (FIG. 26C), while animals treated withMDP demonstrated a striking reduction in the extent of NEC thatdeveloped (FIG. 26D). Administration of MDP alone did not alterintestinal histology (FIG. 26B). These data support the hypothesis thatNOD2 activation with MDP may represent a novel agent to protect againstthe development of experimental NEC.

Discussion. The foregoing experiments provide evidence that the NOD2agonist MDP provides protection from the development of experimental NECin newborn mice, a condition that has been shown to be dependent uponthe activation of TLR4 (Leaphart et al., 2007, J. Immunology179:4808-4820). In terms of understanding the potential mechanism[s]involved, it has been found that MDP limits TLR4 signaling inenterocytes, potentially through an inhibition in TLR4 expression. Thepotential significance of these findings is found in the fact that MDPmay be used to prevent NEC in infants who are at risk for itsdevelopment. The ability to adopt potential preventive strategies ishighlighted by the fact that infants at risk for NEC developmentrepresent a fairly well defined cohort—specifically premature infantsthat have been administered enteral formula. As such, an infant formulathat contains agents that inhibit TLR4 signaling—such as MDP—mayrepresent a novel and exciting therapeutic tool.

What are the potential mechanisms by which MDP may reduce the expressionof TLR4 in enterocytes? It is possible that activation of downstreamtargets of NOD2 by MDP may lead to post-translational modification ofTLR4 that could shorten its half-life. In support of this concept, Yanget al have shown that MDP may alter the ubiquitin state of the TLR4target kinase Rip2, leading to a shortening of its half-life inmacrophages (Yang et al., 2007, J Biol Chem 282:36223-36229); thissuggests that MDP could alter the half-life of TLR4 by similarlyaltering the degree to which TLR4 is ubiquitinated. Alternatively, MDPcould alter the efficiency or rate of transcription of TLR4, throughmechanisms that remain to be defined. MDP may also limit TLR4 signalingthrough a variety of other mechanisms unrelated to the overallexpression of TLR4, such as through effects on the interaction with theadapter protein MyD88, or other downstream targets including IRAK-1.

A link between mutations in NOD2 and inflammatory bowel disease has beensuggested to implicate NOD2 signaling in the suppression of intestinalinflammation (Cho, 2007, Gastroenterology 133:1327-1339). Previousreports have demonstrated that the administration of MDP can limit thedegree of intestinal inflammation in models of ulcerative colitis(Watanabe et al., 2008, J Clin Invest 118:545-559; Yang et al., 2007,Gastroenterology 133:1510-1521; Maeda et al., 2005, Science307:734-738). Although previous reports show protection of MDP in modelsof ulcerative colitis, it is important to point out that ulcerativecolitis and necrotizing enterocolitis are separate and unique diseases:ulcerative colitis affects adults and older children, NEC only affectspreterm or term infants; ulcerative colitis presents with bloodydiarrhea and abdominal pain, NEC presents with progressive and oftenoverwhelming sepsis; ulcerative colitis is a chronic disease that is notfatal; NEC is an acute necrosis of the intestine that is fatal in nearly50% of cases; the pathological hallmark of ulcerative colitis is that ofintestinal inflammation confined to the mucosal lining of the intestinethat never extends into the submucosa, the pathological hallmark of NECis that of mucosal inflammation that always extends into the submucosato extend to the full thickness of the intestine.

9. EXAMPLE 4

The effect of the TLR9 agonist CpG-ODN 5′ TCGTCGTTTTGTCGTTCCTGACGTT 3′(SEQ ID NO:10; referred to herein as CpG-ODN-HS) on the mediator ofactivated TLR4, NFκB, was examined in a murine NEC model (previouslydescribed, above). The effect of CpG-ODN-HS on inflammation in the NECmurine model was also examined as a measured by TNF-α expression, whichis an index of inflammation.

Materials and methods. Phosphorothioated CpG-ODN-HS andoligodeoxynucleotide (ODN) 1826 (TCCATGACGTTCCTGACGTT) (SEQ ID NO:6;referred to herein as CpG-DNA) were synthesized by the University ofPittsburgh DNA synthesis facility. ODNs were confirmed to beendotoxin-free by Limulus assay. NFkB-GFP antibody was obtained fromSanta Cruz and Abcam. The extent of nuclear translocation was determinedin an adaptation of the methodology of Ding and colleagues. In brief, athreshold limit was set based upon the emission signal for the nuclearstain DRAQ5, which therefore defined a nuclear region of interest (ROI).To define a corresponding cytoplasmic region of interest, a circularregion 12 pixels beyond the nucleus was stenciled upon each cell. Theaverage integrated pixel intensity pertaining to the corresponding NFkBemission within the cytoplasmic and nuclear regions was then determinedfor more than 200 cells per treatment group in at least four experimentsper group, using MetaMorph software version 6.1 (Molecular DevicesCorporation, Downingtown, Pa.).

Mucosal TNF-α expression was measured using quantitative real-time PCR(RTPCR). Quantitative real-time PCR in cultured enterocytes andintestinal tissue using the BioRad iCycler (Biorad, Hercules, CA) wasperformed as in (Leaphart, C. L., J. Cavallo, S. C. Gribar, S. Cetin, J.Li, M. F. Branca, T. D. Dubowski, C. P. Sodhi, and D. J. Hackam. 2007. ACritical Role for TLR4 in the Pathogenesis of Necrotizing Enterocolitisby Modulating Intestinal Injury and Repair. J Immunology 179:4808-4820).Oligonucleotide primer pairs used for RTPCR were as follows: forward: 5′CATCTTCTCAAAATTCGAGTGACAA 3′ (SEQ ID NO:14), reverse: 5′CCCAACATGGAACAGATGAGGGT 3′ (SEQ ID NO:15); and forward: 5′TTCCGAATTCACTGGAGCCTCGAA 3′ (SEQ ID NO:16), reverse: 5′AAGGTCTAAGAAGGGACTCCACGT 3′ (SEQ ID NO:17). Gene expression wasnormalized to β-actin expression. Where indicated, gene expression wasassessed on 2.5% agarose gels using ethidium bromide staining. Imageswere obtained with a Kodak (New Haven, CT) Gel Logic 100 Imaging Systemusing Kodak (New Haven, CT) Molecular Imaging software.

Induction of necrotizing enterocolitis. Transgenic mice expressingNFkB-GFP were used for both control and experimental groups. To induceexperimental NEC, 10-14 day-old mice were gavage fed (Similac Advancedinfant formula (Ross Pediatrics): Esbilac canine milk replacer at aratio of 2:1) five times daily, and exposed to intermittent hypoxia (5%O₂, 95% N₂) for 10 minutes using a modular hypoxic chamber(Billups-Rothenberg, DelMar, Calif.) twice daily for 4 days. Animalswere fed 200 microliters per 5 grams of mouse body weight by gavage over2-3 minutes, using a 24-French angio-catheter which was placed into themouse esophagus under direct vision. Samples were harvested at day fourfor analysis. It has been demonstrated that this experimental protocolinduces intestinal inflammation and the release of pro-inflammatorycytokines in a pattern that closely resembles human NEC. Control (i.e.,non NEC) animals remained with their mothers and received breast milk.Where indicated, NEC or breast fed animals of all strains were injectedwith CpG-DNA 1 mg/ml at a daily dose of 1 mg/kg for 4 days prior to. Theseverity of experimental NEC was graded using a previously validatedscoring system from 0 (normal) to 4 (severe). At sacrifice, serum wasobtained by retro-orbital puncture, and terminal ilea was harvested in10% neutral buffered formalin or frozen in liquid nitrogen afterembedding in Cryo-Gel (Cancer Diagnostics, Inc.). Mucosal scrapings wereobtained by microdissection under 20× power, and collected in RNAlater(Qiagen, Valencia, Calif.) for RTPCR analysis. Histological (H&E)analysis if terminal ileum was also performed.

Immuno-analysis. Cryo-Gel (Cancer Diagnostics, Inc.) frozen sections ofterminal ileum were sectioned (4 μm), rehydrated with PBS and fixed with2% paraformaldehyde. Non-specific binding was blocked with 5% bovineserum albumin (BSA). Sections were imaged on an Olympus Fluoview 1000confocal microscope using oil immersion objectives. The ileum sectionswere immunostained with antibodies against the green fluorescent proteinof the NFκB-GFP.

Results

NEC mice exhibited more severe mucosal injury in terminal ileum thanbreast-fed mice (FIGS. 27A, B and G). Histological micrographs ofcontrol and NEC mice treated with CpG-ODN-HS and CpG-DNA demonstrate aninhibition of the NEC pathology in treated animals (FIGS. 27C, D and G).Additionally, mucosal TNF-α expression in the terminal ileum wasincreased in the NEC mice compared to the breast fed control mice,indicating an increase in inflammatory response in the NEC mice.Treating the NEC mice with CpG-ODN-HS or CpG-DNA reduced the level ofexpression of mucosal TNF-α (FIG. 28). Further, activation of NFκB inNEC cells was increased compared to the breast fed controls, but wasreduced when the NEC mice were treated with CpG-ODN-HS (FIG. 29A-D).FIGS. 30A-C show the colocalization of NFκB (i.e., NFκB-GFP) andE-cadherin in ilea cells of control mice (A), NEC mice (B) and NEC micetreated with CpG-ODN-HS.

Various publications are cited herein, the contents of which are herebyincorporated by reference in their entireties.

What is claimed:
 1. A method of treating necrotizing enterocolitis in asubject in need of such treatment comprising administering, to thesubject, an effective amount of a TLR9 agonist oligonucleotidecomprising one or more copy of 5′ GTCGTT 3′ (SEQ ID NO:11).
 2. Themethod of claim 1, where the oligonucleotide further comprises 5′ GACGTT3′ (SEQ ID NO:18).
 3. The method of claim 2, where the oligonucleotidecomprises 5′ TCGTCGTTTTGTCGTTCCTGACGTT 3′ (SEQ ID NO:10).
 4. The methodof claim 1, where the oligonucleotide is orally administered.